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Pakistan is rich in sheep genetic resources. Balkhi, Hashtnagri, and Michni are neighboring sheep populations found in Khyber Pakhtunkhwa province of Pakistan. In this study, we analyzed the genetic structures and bottleneck incidents within these sheep populations using 31 microsatellite DNA markers. Total numbers of 116, 100, and 95 alleles, with average numbers of 3.20, 3.26, and 3.74 alleles per locus were observed, respectively, in Balkhi, Hashtnagri, and Michni population. Mean observed heterozygosity was 0.402 in Balkhi, 0.416 in Hashtnagri, and 0.522 in Michni population. All the three sheep populations showed significantly high inbreeding. Michni population was found to be in mutation drift equilibrium, showing the absence of genetic bottleneck. The data of Balkhi and Hashtnagri indicated the presence of genetic bottleneck in these populations. These results suggest a moderate level of genetic diversity within Michni population that may be useful for breed improvement programs. Hashtnagri and Balkhi populations having low within breed genetic variability may contain some valuable characteristics that need to be conserved.

To combat the ever-increasing threat of wheat yellow rust worldwide, understanding of the pathogen (Puccinia striiformis) population biology is indispensable. Molecular markers, particularly microsatellites, have been reported to be important tools for deciphering pathogen population structure, invasion sources, and migration history. The utility of these DNA-based markers and sequencing has been increased by the direct DNA extraction from infected leaves with subsequent multiplex-based SSR genotyping. In this chapter we describe the protocol for direct DNA extraction and its genotyping with microsatellite markers in multiplex reactions. We describe the procedure for allele scoring, and various troubles faced during microsatellite scoring and potential solutions for them.

Knowledge about genetic diversity among Brassica napus cultivars developed for many growing regions and their possible use as potential inbred lines for hybrid seed production is limited. We studied the genetic diversity and relationships among B. napus accessions using Sequence Related Amplified Polymorphism (SRAP) markers, which preferentially amplify open reading frames. A total of 60 spring-type B. napus accessions were screened using 20 SRAP primers, which revealed 162 polymorphic fragments with an average of eight markers per primer combination. Genetic similarity estimates ranged from 40 to 100, which indicated sufficient diversity among the accessions. The majority of the accessions were uniquely identified by the markers with the exception of near-isogenic inbred lines. Cluster analysis displayed five major groups. The first major cluster comprised 23 accessions mostly of Australian origin, whereas the second cluster included 13 accessions mostly of Canadian origin. The accessions in the first and second clusters were identified as maintainers of cytoplasmic male sterility. The two restorer lines R-111 and R-101 along with their corresponding backcross progeny constituted the third cluster. Scandinavian cultivars made the fourth separate cluster. One cultivar Salam and its respective inbred line were the most divergent lines. Variations in the number of markers between open-pollinated cultivars and their respective selfed inbred lines were also observed. The clustering pattern mostly supported their respective pedigree and characteristic traits. Genetic diversity in genetically distinct groups in the tested maintainer and restorer lines can be exploited for hybrid development in B. napus.

Mycoplasma infections are of great concern in avian medicine, because they cause economic losses in commercial poultry production of Pakistan. Timely and efficient diagnosis of the mycoplasma is needed in order to practice prevention and control strategies. A duplex Polymerase Chain Reaction (PCR) was optimized for simultaneous detection of two pathogenic species of avian mycoplasmas. Two sets of oligonucleotide primers specific for Mycoplasma gallisepticum and Mycoplasma synoviae, were used in the test. The developed assay exhibited high sensitivity and 100% specificity for the simultaneous detection of mycoplasma species prevalence in Pakistan.

Balkhi, Michni and Hashtnagri populations are the three fat-tailed phenotypes native to Khyber Pakhtunkhwa Province of Pakistan, harbouring the central valley. Their distribution, breeding trend and performances were evaluated through a survey using a pre-tested questionnaire during August, 2008. Out of the total populations, 70.7% were mixed flocks and 5.8%, 3.1% and 9.2% were purebred flocks belonging to Balkhi, Hashtnagri and Michni populations, respectively. Sheep flocks were reared under three different production systems, i.e. sedentary, transhumant and nomadic. Nomads keep Balkhi and are sole responsible for their development as a mutton breed. They practise autumn breeding only, for which they select the heaviest ram/s available within the community. Sedentary and transhumant shepherds had mixed flock, who were constantly replacing their native breed stock, i.e. Michni and Hashtnagri through outcrossing with Balkhi. The size of Balkhi and Michni populations warrants no immediate threat to their existence and diversity. However, in case of increased popularity of crossbreeding, Michni could pose threats of extinction. Hashtnagri on the other hand can be declared as breed at risk. Age at first lambing was significantly lower in Hashtnagri ewes, followed by Michni, crossbred and Balkhi. Hashtnagri ewes outperformed other populations in lambing twice per annum.

Buffaloes usually maintain higher body condition and do not produce milk at the cost of their own body reserves under tropical conditions. The mobilization of body reserves for fulfilling the demands of lactation has been extensively studied in dairy cows while limited work is available on this aspect in dairy buffaloes. Therefore, the present study was conducted to examine variations in milk fatty acid profiles with body condition in Nili-Ravi buffaloes. A total of 24 Nili-Ravi buffaloes within 60 days after parturition, were selected from a private dairy farm in the district of Peshawar. All animals consumed the same diet during the experimental period. A total of 576 raw milk samples were collected for laboratory analysis. The study continued up to 6 months during 2008. Body condition score (BCS), milk yield and composition were recorded once a week. Means for milk fatty acid profile were compared for various levels of BCS. The mean milk yield and fat content were 9.28 kg/d and 5.36%, respectively. The total saturated fatty acids (SFA) were 64.22 g/100 g and the unsaturated fatty acids (UFA) were 35.79 g/100 g. Of the SFA the highest amount was recorded for , followed by , and . The total sum of hypercholesterolemic fatty acids (HCFA, , and ) was 43.33 g/100 g. The concentrations of UFA were greater for moderate BCS followed by poor and highest BCS while SFA showed the opposite trend. The correlation analysis showed that milk yield was negatively affected by BCS and milk fat positively affected, though non-significantly. The present study suggests that Nili-Ravi dairy buffaloes produce similar milk to dairy cows regarding availability of cardioprotective fatty acids, with the highest concentration of . Two HCFA ( and ) were associated with higher body condition. Buffaloes with moderate body condition yielded milk containing healthier fatty acids.

Exposure of plants to long periods of water scarcity, mainly in arid and semi-arid regions, is one of the major reasons for over 50% reduction in average yields. Proper management of Brassica napus to enhance its ability to survive under drought and high temperature stress at early growth stages, besides development of tolerant genotypes, could improve its production in the rainfed areas. In the present study, we report the interactive effects of Ca 2+ , K + and N supplementation and water availability on the fresh biomass, N and C content, as well as C/N ratio of crop plants. Exposure to water stress significantly reduced the fresh biomass, Nitrogen (N), Carbon (C) content and the C/N ratio. Ca 2+ and K + supplementation before drought positively affected fresh biomass by stimulating N uptake and C assimilation. However, the C/N ratio was reduced after supplementation with Ca 2+ and K + . The N supplementation before drought imposition, though enhanced the N uptake, but the excessive damage to cell membranes and electrons leakage from Electron Transport Chain (ETC) during photosynthesis resulted in a decrease in C assimilation. Consequently, there was a decrease in the C/N ratio in seedlings exposed to drought after supplementation with N. Key words : Drought, nutrients supplementation, carbon, nitrogen, C/N ratio.

Supply of adequate moisture is one of the most important factors limiting agricultural extension and productivity. Ca 2+ and K + are both essential plant nutrients required in a number of developmental and metabolic processes. Both of these nutrients play an important role in ameliorating drought stress in crop plants. This experiment was designed to study whether exogenous application of Ca 2+ and K + before the drought could enhance the potential of plants to survive under limiting water conditions. Brassica napus L. cv Bulbul-98 seedlings were exposed to drought stress for 10 days after exogenous application of different concentrations of Ca 2+ , K + and N through irrigation water. Exogenous Ca 2+ and K + application was associated with increased relative water content, membrane stability and chlorophyll content in seedlings exposed to water deficit. Though the quantity of Pro and activities of APX (EC: 1.11.1.11) and CAT (EC: 1.11.1.6) were increased after K + application, they were strongly induced in seedlings supplemented with Ca 2+ in a dose dependent manner. Increasing quantity of N application, however, had a negative effect on these parameters. Key words : Drought, Ca 2+ , K + , AOS enzymes, proline, chlorophyll.

Callus induction from cotyledon explants was studied in indigenous chick pea ( Cicer arietinum L.) cultivars KK-1 and Hassan-2K on MS and B 5 media containing different combinations and concentrations of growth regulators. Different MS and B 5 callusing media containing varying level of 2, 4-D (2 and 4 mg/l), NAA (0.50 and 1 mg/l), BAP (5 and 10 μM) and their combinations were tested for callus induction response. Percent callus and callus fresh weight (g) were recorded after two and four weeks of culture for both genotypes. For KK-1 cultivar, the maximum callus frequency (71 and 97%) followed by (65 and 96%) were observed on 4 mg/l 2,4-D+5 μM BAP in MS and 4 mg/l 2,4-D in B 5 media, respectively after two and four weeks of culture. Similarly, the highest callus fresh weight (0.411 and 0.787 g) were also recorded for MS+4 mg/l 2,4-D+5 μM BAP in contrast to B 5 where the highest callus weight (0.401 and 0.693 g) was achieved on 4 mg/l 2,4-D only. In Hassan -2K, the highest callus % (68 and 96) and fresh weight (0.572 and 0.821 g) were recorded on MS+4 mg/l 2,4-D+0.50 mg/l NAA after two and four weeks of culture, respectively. In B 5 medium, 2,4-D+BAP combination produced average callus induction response for both cultivars. Key words : Callus induction, chick pea cotyledons, growth regulators.

Background Hepatitis C virus (HCV) genotype 3a is known to show comparatively better response to combination therapy than genotype 1 and 4. Mutations within NS5A gene of HCV have earlier been implicated with response to interferon (IFN) therapies in chronic HCV patients among various populations. As response to therapy are available in different populations because of the ethnic and viral factors and there was no study available on the phenomenon of resistivity to IFN. Results Chronic HCV 3a infected Pakistani patients were kept on IFN-α and ribavirin therapy for six months. NS5A gene of HCV was amplified and sequenced in the case of all the patients prior to therapy and the sequences were analysed for mutations. Out of the total 27 patients, 20 (74.07%) were observed with sustained virological response (SVR), 4 (14.81%) patients were non responder (NR) while 3 (11.11%) patients exhibited in end of treatment response (ETR). Three (3/20) (15%) SVR patients and two (2/3) ETR patients had mutations (ranging from I-V amino acids) within the NS5A ISDR regions. While the rest of the SVR patients (85%) and the NR had no mutations at ISDR region when compared with HCV K3a ISDR. Conclusions Mutations within the NS5A gene of HCV 3a genotype may not influence the outcome of combination therapy in Pakistani populations.

Estimation of genetic diversity of Brassica germplasm provides the basis for rapeseed/mustard genetic improvement. Studies were undertaken to estimate the genetic diversity of 30 lines of Brassica napus using Randomly Amplified Polymorphic DNA (RAPD) primers. A total of 30 B. napus genotypes of local and exotic origin were characterized using molecular markers. Four RAPD primers were used to estimate the genetic distances among the genotypes in all the possible combinations. The genetic diversity study revealed different levels of genetic polymorphism for RAPD primers GLA05, GLA07, GLA09 and GLA 10, resulting in amplification of 5.7, 3.5, 3.1 and 5.4 scorable bands (loci) per genotype, respectively. Individual genetic distances observed among B. napus genotypes ranged from 6.5 to 51%. Bivariate data matrix was generated and genetic distances were calculated using Unweighted Pair Group of Arithmetic Mean (UPGMA) procedure. The UPGMA cluster analyses revealed maximum genetic dissimilarity for 8966-1 and 8969-1 genotypes, closely followed by Ganyou-5, 89127-1, 89111-2 and Mlep-048. It is recommended that among the thirty B. napus genotypes, genetically distinct lines pointed out in the present study, should be used in future breeding programs for improvement of Brassica napus.